Project Summary Rejection remains a major hurdle to long-term transplant survival. In fact, from a clinical standpoint current immunosuppression protocols are fantastic in short-term transplant survival, but do little in promoting long- term graft outcomes. Thus, there is a pressing need now to unravel effector programs that may develop under broad immunosuppression conditions that can mediate graft loss. We recently discovered that unlike other Th subsets, Th9 cells are prominently induced under unusual circumstances in that they may be especially relevant to graft loss in immunosuppressed hosts. They are best induced when the default Th1/Th2 programs are inhibited (immunosuppression), and require OX40-induced formation of super enhancers at Il9 locus (not specific transcription factors), and induction of Th9 cells is often at the cost of Foxp3+ Tregs. Based on the unexpected potency of OX40 in Th9 induction, as well as the unexpected mode of actions of OX40 stimulation, we propose and test a unifying hypothesis in this proposal, namely- suppression of the default T cell programs in an immunosuppressive milieu allows OX40 the opportunity to support alternative Th9 programs in triggering rejection of otherwise tolerant grafts. We propose 3 Aims in this proposal. Aim 1 focuses on identifying novel mechanisms by which OX40 promotes massive Th9 cells, examining formation, structure, and activities of super enhancers at Il9 locus. Aim 2 investigates how OX40 redirects Foxp3+ Tregs to Th9 cells, focusing on the newly identified Foxp3 repressors in shutting down Foxp3 expression and whether Foxp3 may disrupt IL-9 super enhancer formation. Aim 3 examines the impact of Th9 cells in transplant survival, testing whether Th9 cells mediate graft loss under tolerizing therapies. We believe that the proposed studies will unravel new mechanisms of graft injury in tolerizing hosts and findings from these studies will have broad impacts on other immune-mediated diseases as well, including cancer immunotherapies and autoimmune diseases.